The goal of this proposal is to understand the role of the anticodon loop in tRNA function. The approach is synthetic. We have developed efficient procedures to cleave the polynucleotide chain in the anticodon loop, remove one or more nucleotides, insert other nucleotides and reseal the tRNA to produce a varient molecule with a defined chemical change in the anticoden. The substrate specificity of the enzymes (polynucleotide kinase and RNA ligase) used for this procedure are sufficiently broad that an enormous variety of base and sugar modifications can be inserted into the polynucleotide chain. Thus, a high degree of synthetic control over a crucial portion of the tRNA sequence has been achieved. We plan to use this technology to prepare varient tRNAs with different modifications in the anticodon loop to examine two aspects of tRNA function. First, we want to evaluate the extent to wich the anticodon loop is part of the "recognition" sites of yeast phenylalanine and tyrosine synthetases. Thus, the kinetics of the aminoacylation reaction will be determined for different modified tRNAphe and tRNAtyr. The second area of interest is the codon-anticodon interaction of the ribosome. Anticodon varients of both tRNAS will be examined in several different types of assays to evaluate how effective they are in the decoding process. A secondary goal of this proposal is to evaluate the generality of the enzymatic nucleotide substitution procedure. This will be done by attempting to alter a structured region of tRNA where the reactions are expected to be more difficult. This approach is expected to be very useful for structure-function studies in other RNA molecules.